EP1572230B1

EP1572230B1 - Placental alkaline phosphatase to control diabetes

Info

Publication number: EP1572230B1
Application number: EP03796758A
Authority: EP
Inventors: Zoltan Kiss
Original assignee: Zoltan Laboratories
Current assignee: Zoltan Laboratories
Priority date: 2002-12-12
Filing date: 2003-12-05
Publication date: 2010-09-08
Anticipated expiration: 2023-12-05
Also published as: US7501116B2; AU2003298036A1; WO2004054609A1; EP1572230A1; US20060034818A1

Abstract

The present invention provides methods for using human placental alkaline phosphatase or an active derivative to reduce blood glucose level in a mammal. Treatment regimens provided by the invention may be used to control Type 1 and Type 2 forms of diabetes in humans. The methods and treatment regimens may be effective to maintain the human's blood glucose level below about 10 mM, and preferably within the normal range of 4 mM to 7 mM. The methods and treatment regimens may be used in combination with administration of known anti-diabetic medicaments. Also provided by the invention is a method for inducing weight loss or reducing an expected weight gain caused by or associated with obesity or Type 2 diabetes. The invention further provides a preparation for administration to a human, the preparation comprising homogeneous purified human placental alkaline phosphatase in a physiologically acceptable carrier.

Description

Technical Field

The present invention relates to methods for using human placental alkaline phosphatase, an enzyme produced by human placenta during pregnancy, to reduce blood glucose level in a mammal. Treatment regimens provided by the invention may be used to control Type 1 and Type 2 forms of diabetes in humans.
Significant recent changes in human behavior and lifestyle as well as the human environment have resulted in the escalation of diabetes during the last decades. Diabetes is a disease characterized by elevated levels of blood plasma glucose, or hyperglycemia. Hyperglycemia, if uncontrolled, can lead to other complications, such as blindness, kidney disease, heart disease, stroke, nerve diseases, circulatory disorders, and impotence in males. Diabetes is a chronic disease with diverse pathologic manifestations, and is accompanied by lipid metabolism and cardiovascular disorders as well as glycometabolism disorders.
Diabetes mellitus is a heterogeneous group of disorders characterized by high blood glucose (sugar) levels. There are two main types of diabetes. Type 1, or insulin-dependent diabetes, results from a deficiency of insulin due to autoimmunological destruction of the insulin-producing pancreatic β-cell islets [Bell, G.I. and Polonsky, K.S., "Diabetes mellitus and genetically programmed defects in β-cell function." Nature 414, 788-791 (2001); Mathis, D., Vence, L. and Benoist, C., "β-cell death during progression to diabetes." Nature 414, 792-798 (2001)]. People with Type 1 diabetes must take exogenous insulin for survival to prevent the development of ketoacidosis.
In Type 2 diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), muscle, fat, and liver cells are resistant to the actions of insulin. Furthermore, compensatory mechanisms that are activated in β-cells to secrete more insulin to maintain blood glucose levels within a normal physiological range fail to function properly. Type 2 diabetes accounts for about 90% of all diabetes [Saltiel, A.R., "New perspectives into the molecular pathogenesis and treatment of )]. Type 2 diabetics are often prescribed blood glucose-lowering sulfonylurea-based or-derived drugs, which are associated with the stimulation of insulin production in the pancreatic β-cells. Alternatively, patients suffering from Type 2 diabetes may also be prescribed biguanide-based or -derived drugs, which are associated with increasing a patient's sensitivity to insulin.
Diabetes already afflicts an estimated 6% of the adult population in Western society, and its worldwide frequency is projected to grow by 6% per annum, potentially reaching a total of 200-300 million cases in 2010 [Zimmet, P., Alberti, K.G.M. and Shaw, J., "Global and societal implications of the diabetes epidemic." Nature 414, 782-787 (2001)]. The main forces driving this increasing incidence are sedentary lifestyle and a staggering increase in obesity.
Diabetes is a potentially very dangerous disease because it is associated with markedly increased incidence of coronary, cerebral, and peripheral artery disease. As a result, patients with diabetes have a much higher risk of myocardial infarction, stroke, limb amputation, renal failure, or blindness. Atherosclerotic cardiovascular disease is responsible for 80% of diabetic mortality and more than 75% of all hospitalizanons for diabetic complications [Moller, D.E., "New drug targets for )]. Recent evidence indicate that hyperglycemia leads to overproduction of superoxide accounting for vascular damage, which, in turn, underlies most diabetic complications [Brownlee, M., "Biochemistry and molecular cell biology of diabetic complications." Nature 414, 813-820 (2001); Ho, E. and Bray, T.M., "Antioxidants, NFκB activation, and diabetogenesis." Proc. Sac. Exp. Biol. Med. 222, 205-213 (1999)].
Despite large variations in carbohydrate intake with various meals, blood glucose normally remains in a narrow range between 4 and 7 mM in non-diabetic individuals. Such tight control is regulated by the balance among three major mechanisms, i.e. (i) glucose absorption from the intestine, (ii) glucose production by the liver, and (iii) uptake and metabolism of glucose by the peripheral tissues, mainly the skeletal muscle and fat tissue. In skeletal muscle and fat tissue, insulin increases the uptake of glucose, increases the conversion of glucose to glycogen, and increases conversion of glucose to fat (mainly triglycerides). In the liver, insulin inhibits the release of glucose from glycogen. Insulin is the only known hormone which can regulate all three mechanisms required to maintain the blood glucose level in the normal range [Saltiel, A.R. and Kahn, C.R., "Insulin signaling and the regulation of glucose and lipid metabolism" Nature 414, 799-806 (2001)].
At present, the only established available treatment for severe Type 1 diabetes is daily (often multiple) insulin injection. Apart from the inconvenience of the injection procedure, several adverse effects may accompany insulin treatment, including occasionally severe hypoglycemia (lower than normal blood glucose level) and weight gain; unfortunately, this latter side effect can make the target tissues even more resistant to the actions of insulin [U.K. Prospective Diabetes Study Group, "U.K )]. On an experimental basis, clinical islet transplantation is also gaining acceptance, particularly for patients with hypoglycemic awareness [Ryan, E.A., Lakey, J.R.T., Paty, B.W., Imes, S., Korbutt, G.S., Kneteman, N.M., Bigam, D., Rajotte, R.V. and Shapiro, A.M.J., "Successful islet transplantation: Continued insulin reserve provides long-term glycemic control." Diabetes 51, 2148-2157 (2002)]; however, this method is still far from established. Apart from the problem that it is very difficult to adjust insulin production to meet the patient's needs, immunosuppressants, which are used to prevent rejection of the transplanted tissue, can exert potent and undesirable side effects. It is obvious that even partial replacement of insulin with a safer and effective agent that does not require a complicated surgical procedure would greatly benefit Type 1 diabetic patients.
In Type 2 diabetes, an aggressive control of hyperglycemia by medication is essential; otherwise this disease will progress into the even more dangerous Type 1 diabetes. Several drugs in five major categories, each acting by a different mechanism, are available for this purpose [Moller, D.E., "New drug targets for )]:

(A) Insulin secretogogues, including sulphonylureas (e.g., glipizide, glimepiride, glyburide) and meglitinides (e.g., nateglidine and repaglinide), enhance secretion of insulin by acting on the pancreatic β-cells. While this therapy can decrease blood glucose level, it has limited efficacy and tolerability In addition, it causes weight gain and often induces hypoglycemia. Finally, patients often become refractory to this treatment.
(B) Biguanides (e.g., metformin) are thought to act primarily by decreasing glucose production in the liver. Biguanides often cause gastrointestinal disturbances and lactic acidosis, which limits their use.
(C) Inhibitors of α-glucosidase (e.g., acarbose) decrease absorption of glucose from the intestine. These agents also often cause gastrointestinal disturbances.
(D) Thiazolidinediones (e.g., pioglitazone, rosiglitazone) act on a specific receptor (peroxisome proliferator-activated receptor-gamma (PPARγ)) in the liver, muscle and fat tissues. They regulate lipid metabolism and thus enhance the response of these tissues to the actions of insulin. Frequent use of these drugs may lead to weight gain and may induce edema and anemia.
(E) Insulin is used in more severe cases, either alone or in combination with the above agents.

Because each agent that is being used in the medical treatment of diabetes has either significant side effects or causes weight gain (which, in turn, further impairs the actions of insulin), or both, newer approaches to control Type 2 diabetes are desperately needed. An effective new treatment would meet the following criteria: (a) it would not have significant side effects including induction of hypoglycemia; (b) it would not cause weight gain; (c) it would at least partially replace insulin by acting via mechanism(s) that are independent from the actions of insulin; (d) it would desirably be metabolically stable to allow less frequent (e.g., once per week) usage; and (e) it would be usable in combination with tolerable amounts of any of the categories of drugs listed above.
Placental alkaline phosphatase (PALP) is a member of the alkaline phosphatase group of enzymes that hydrolyze phosphate-containing compounds at alkaline pH. Mature PALP is a dimer of two identical glycosylated subunits. A primary source of PALP is human placenta, which synthesizes this enzyme during pregnancy so that toward the end of third term the enzyme's level in the placenta tissue and matemal/fetal blood becomes very high. Therefore, it is very unlikely that human PALP exerts toxic or pathological effects in human tissues. Subunits of human placental alkaline phosphatase have an approximate molecular weight of 66 kDa, as determined by gel electrophoresis.
A determination of an in vivo half-life for human PALP was reported in 1965 [Clubb, J.S., Neale, F.C. and Posen, S., "The behavior of infused human placental alkaline phosphatase in human subjects." J. Lab. & Clin. Med. 66, 493-507 (1965)]. In human subjects, injected PALP is reported to remain remarkably stable in the circulation, with an estimated biological half-life of about 7 days. In the reported experiments, PALP was injected as a minor constituent in a mixture of PALP and albumin obtained by extraction, without further purification. The authors reported that PALP up to serum concentration of 975 "King-Armstrong" (KA) units appeared metabolically inert, and hypothesized that PALP performs no measurable physiological function in circulation.
The physiological function of PALP has been unknown until recently, when Kiss and his co-workers discovered that, in human fetus and mouse embryo fibroblasts, the enzyme functions both as a growth factor and a promoter of survival in serum factor-deficient culture medium [She, Q.-B., Mukhezjee, J J., Huang, J.-S., Crilly, K.S. and Kiss, Z., "Growth factor-like effects of placental alkaline phospharase in human and mouse embryo fibroblasts." FE BS Lett. 469, 163-167 (2000); She, Q.-B., Mukherjee, J.J., Chung, T. and Kiss, Z., "Placental alkaline phosphatase, insulin, and adenine nucleotides or adenosine synergistically promote long-term survival of serum-starved mouse embryo and human fetus fibroblasts." )].
WO 02/072 136 discloses that placenta alkaline phosphatase interacts synergistically with growth factors and corresponding serum factors to stimulate the proliferation of adult fibroblast cells. Brock et al Brit. J. Obstetr. gyn, vol. 95, no.1, 1988 p. 79-83 pertains to the measurement of AP in maternal plasma as an indicator of subsequent low birthweight outcome.

Brief Description of the Drawings

Fig. 1 shows a picture of a gel separation, demonstrating that homogeneous purified PALP used for the experiments described in Examples 21, 22, and 24 does not contain any contaminating protein.
Fig. 2 demonstrates that in BDF1 male fasting mice, administration of a preparation comprising commercial PALP prior to a glucose load substantially reduced an increase in blood glucose level.
Fig. 3 demonstrates that in BDF1 male mice, intraperitoneal application of streptozotocin increased blood glucose level about 9-fold in three days(■), and that prior administration of a preparation comprising commercial PALP greatly reduced such increase in glucose level (◆).
Fig. 4 demonstrates that in BDF1 male mice, intraperitoneal application of streptozotocin considerably decreased body weight over an eight day period (■), and that prior administration of a preparation comprising commercial PALP partially prevented the decrease in body weight (◆).
Fig. 5 indicates that in differentiated L1 adipocyte cells, commercial PALP stimulates cellular uptake of D-[¹⁴C]glucose at various levels of glucose and serum in the incubation medium.
Fig. 6 demonstrates that in differentiated L1 adipocyte cells, commercial PALP stimulates the cellular synthesis of glycogen from D-[¹⁴C]glucose at various compositions of the incubation medium.
Fig. 7 demonstrates that in differentiated L1 adipocyte cells, commercial PALP stimulates the cellular synthesis of lipids from D-[¹⁴C]glucose at various compositions of the incubation medium.
Fig. 8 demonstrates that in differentiated L6 muscle cells, both 1 U/mL commercial PALP and 50 nM insulin enhance the uptake and metabolism of D-[¹⁴C]glucose after treatments for 6 hours; PALP and insulin in combination had approximately additive effects.
Fig. 9 shows a picture of a gel separation demonstrating that digestion of human PALP with bromelain results in the formation of a major fragment and several smaller fragments, concomitant with the disappearance of native PALP enzyme.
Fig. 10 demonstrates that in differentiated L1 adipocyte cells, a product of the digestion of human PALP by bromelain enhances the synthesis of glycogen, but not lipids, from D-[¹⁴C]glucose, relative to native PALP. Bromelain did not affect the total cellular level of D-[¹⁴C]glucose, but slightly enhanced glycogen and lipid synthesis.
Fig. 11 demonstrates that in differentiated L1 adipocyte cells, treatment with either commercial PALP or insulin for 6 hours decreased the amount D-[¹⁴C]glucose in the incubation medium, indicating that both agents enhance cellular uptake of the radiolabeled glucose.
Fig. 12 indicates that in differentiated L1 adipocytes, 1 U/mL commercial PALP is more effective than 10 nM insulin in enhancing cellular uptake of D-[¹⁴C]glucose, as well as metabolism of the radiolabeled glucose to form glycogen and lipids, at both 2 hours and 6 hours after treatment.
Fig. 13 demonstrates that in differentiated L1 adipocytes, 1 U/mL commercial PALP is somewhat less effective than 50 nM (supraphysiological) insulin in stimulating uptake and metabolism of D-[¹⁴C]glucose, at both 2 hours and 6 hours after treatment.
Fig. 14 demonstrates that in mouse embryo NIH 3T3 fibroblasts, commercial PALP is more effective than insulin in stimulating uptake and metabolism of D-¹⁴C]glucose, and that insulin does not modify the effect of PALP when used in combination with PALP.
Fig. 15 indicates that in differentiated L1 adipocytes, preparations made from different batches of commercial PALP (each preparation containing 1.5 U/mL) have effects on the loss of D-[¹⁴C]glucose from incubation medium that include some variability which appears to be larger than the inter-experimental error. Each of the preparations was somewhat less effective than 50 nM insulin.
Fig. 16 indicates that in differentiated L1 adipocytes, preparations made from different batches of commercial PALP have effects on the synthesis of glycogen and lipids from D-[¹⁴C]glucose that include some variability.
Fig. 17 demonstrates that in differentiated L1 adipocytes, homogeneous purified PALP is somewhat less effective than insulin in reducing the amount of D-[¹⁴C]glucose in incubation medium.
Fig. 18 demonstrates that in differentiated L1 adipocytes, homogeneous purified PALP is somewhat less effective than insulin in enhancing uptake and metabolism of D-[¹⁴C]glucose.
Fig. 19 demonstrates that in differentiated L1 adipocytes, an antibody against PALP is capable of inhibiting all effects of homogeneous purified PALP on the uptake and metabolism of D-[¹⁴C]glucose.

Detailed Description of the Invention

The observation of the stimulatory effects of PALP on cell proliferation, described above, led to the experiments described in the Examples herein. In a first set of experiments, the effects of PALP on blood glucose level were determined by glucose tolerance tests in mice, as well as in models for Type 1 and Type 2 diabetes. In a second set of experiments, the effects of PALP on cellular glucose uptake in appropriate model systems for fat tissue (differentiated L1 adipocytes) and skeletal muscle (differentiated L6 muscle cells) were observed.
The results of the sets of experiments demonstrate that PALP has powerful insulin-independent stimulatory effects on glucose uptake and metabolism in vitro, and corresponding significant glucose lowering effects in vivo, without pathological effects and without causing weight gain. The data, combined with other observations including a) that high levels of PALP appear to exert only positive physiological effects in pregnant women, and b) that the enzyme is very stable in the circulation, indicate thai PALP is likely to provide effective, safe, and sustained control of hyperglycemia in diabetic patients. Accordingly, the invention includes the use of PALP as an effective and safe blood glucose-reducing agent, and as an effective agent in a treatment regimen for the treatment of diabetes.

Method for Reducing Blood Glucose Level

In one embodiment, the present invention provides a method of reducing blood glucose level in a mammal, comprising the step of administering a therapeutically effective amount of purified human placental alkaline phosphatase (PALP), or an active derivative selected from fragments of PALP that demonstrate efficacies similar or more effective than native PALP. The term "therapeutically effective amount" in this specification and in the claims indicates a dosage that is effective in, or is targeted to, either attaining a desired level of blood glucose or maintaining a desired level of blood glucose over an appropriate time window.
In the practice of the methods of this embodiment, the mammal may be a human. The method maybe effective to reduce a human's blood glucose level to below about 10 mM, and preferably into the normal range of about 4 mM to about 7 mM. The method maybe used on a diabetic human, and maybe used in combination with administration of an anti-diabetic medicament.
The method may also be used for preventing the onset of Type 2 diabetes, for a human that is at risk for the development of diabetes. In particular, the method may be appropriate for a human that exhibits a chronically elevated blood glucose level, but that has not yet been diagnosed with diabetes. Furthermore, the method maybe useful for preventing a progression from Type 2 diabetes to Type 1 diabetes. This is because the persistently high blood glucose levels associated with Type 2 diabetes eventually leads to the destruction of insulin-producing β-cells in the islets of Langerhans, resulting in an insulin deficiency.

The Active Component

The active component in the methods and compositions of the present invention is human PALP, or an active derivative as defined above. As used herein, the term "PALP" and the phrase "human PALP" are used interchangeably to refer to human placental alkaline phosphatase.
As is demonstrated by the examples herein, whole PALP enzyme in its native state is not required to achieve a glucose-reducing effect. Active derivatives of PALP are therefore suitable for the practice of the present invention. For example, digestion of PALP by a protease, such as bromelain, may provide an active derivative. Likewise, one who is skilled in the art may synthesize or develop an active derivative that is a smaller fragment of a PALP sequence which demonstrates efficacy similar to that of native PALP enzyme. In the practice of the present invention, it is envisioned that fragments of PALP may be similarly effective or even more effective than the native PALP enzyme, and are each considered to be active derivatives.
Human PALP in solid form is available commercially from Sigma Chemical (St. Louis, Missouri), for example (Sigma catalog number P3895; CAS Registry Number 9001-78-9). Another commercial source of human PALP is Calbiochem (San Diego, California; catalog number 524604).
Human PALP may also be obtained by extraction from placental tissue. For example, a partially purified preparation may be obtained by butanol extraction of homogenized placenta. Other methods of extraction from placental tissue are also suitable.
Human PALP, or an active derivative as defined above, may also be obtained by chemical synthesis using conventional methods. For example, solid phase synthesis techniques may be used to obtain PALP or an active derivative. Recombinant methods of obtaining quantities of PALP are also suitable, as is known in the art.
The methods and treatment regimens described rein include administration of purified human placental alkaline phosphatase, or said active derivative. In the practice of the present invention, PALP or an active derivative is generally administered in the form of a preparation comprising the PALP enzyme or said active derivative. The term "preparation" as used herein means a composition comprising purified human PALP or said active derivative dissolved or dispersed in a carrier. In a preparation for administration to a human, the carrier should be a physiologically acceptable carrier.
The term "purified" is used herein to encompass compositions that are obtained from a starting material by one or more purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enhance the concentration of active agent relative to the starting material. The term "purified" also encompasses compositions that contain a significant quantity of active agent in relation to impurities, whether obtained by a purification process or not. The term "purified" should not be construed to connote absolute purity.
The phrase "purified human placental alkaline phosphatase" therefore includes compositions such as a partially purified human PALP available from Sigma Chemical, which was used as a starting material for the experiments described in the following Examples.
A purified human PALP composition may also be obtained, for example, by a purification procedure described elsewhere [She, Q.-B., Mukherjee, J.J., Huang, J.-S., Crilly, K.S. and Kiss, Z., "Growth factor-like effects of placental alkaline phosphatase in human and mouse embryo fibroblasts." FE BS Lett. 469, 163-167 (2000)].
The term "homogeneous" is used herein to indicate a composition that yields a single protein band in an electrophoretic gel separation, such as by the SDS-PAGE technique described in Example 1. The phrase "homogeneous purified human placental alkaline phosphatase" therefore includes compositions that yield a single band for PALP enzyme in an electrophoretic separation. A homogeneous purified human PALP may be obtained, for example, by the purification procedures described in Example 1.
A separate consideration is the degree of purity that is required for PALP material that is to be used in a preparation for administration to a human. An advantage of using a preparation comprising highly purified or homogeneous PALP in the methods and treatment regimens of the present invention is that possible side effects caused by contaminating proteins will not be an issue. However, less extensively purified PALP preparations, such as that comprising the commercially available human PALP from Sigma, may also be used so long as safety can be demonstrated. Since each additional purification step results in significant loss of the enzyme, using less purified PALP material for PALP preparations would be more cost-effecave.

Administration of PALP

Since human PALP is a relatively large protein, and its actions appear to involve the adipose tissue and muscle, systemic administration is an appropriate mode of administration.
By way of example, systemic administration may include administration of a PALP-containing preparation. For administration to a human, purified human PALP is dissolved or dispersed in physiological saline or in another physiologically acceptable carrier, or enclosed in liposomes such as immunoliposomes, or other delivery systems or formulations as are known to the art, to produce a PALP-containing preparation.
For example, one suitable PALP preparation for the practice of the present invention comprises purified human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 10 mg/mL. Another suitable PALP preparation comprises purified human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 30 mg/mL.
A PALP preparation may be administered by injection, for example. A PALP preparation may be administered via intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, intramuscular injection, or any other mode of delivery that ensures appropriate distribution and relative stability of the enzyme in the body.

In one embodiment of the method, purified human placental alkaline phosphatase, or said active derivative, is administered to a human. A common way to express a suitable dosage is grams of active agent per square meter of body surface area for the subject. Several formulas are known for estimating a human subject's body surface area, based on the human's height (in cm) and mass (in kg). Table 1 lists a variety of known formulas for estimating body surface area (BSA) proposed by researchers. Other suitable formulas may likewise be employed.

Table 1. Formulas for estimating body surface area (BSA).

Author(s)	BSA formula
Du Bois and Du Bois	BSA (m²) = Mass(kg)^0.425	x Height(cm)^0.725	x 0.007184
Gehan and George	BSA (m²) = Mass(kg)^0.51456	x Height(cm)^0.42246	x 0.02350
Haycock	BSA (m²) = Mass(kg)^0.5378	x Height(cm)^0.3964	x 0.024265
Mosteller	BSA (m²) = Mass(kg) ^0.5	x Height(cm) ^0.5	x 0.016666

Preferably, a dosage of about 0.2 to about 3 g/m² of purified human PALP or said active derivative is administered to the human; more preferably, a dosage of about 0.2 to about 1.0 g/m² is administered to the human.
The human may have an elevated blood glucose level before administration of purified human PALP. By "elevated," it is meant that the human's blood glucose level is greater than the normal range of about 4 to about 7 mM. Systemic administration of PALP preferably reduces blood glucose level in a human to below about 10 mM, more preferably to below about 8 mM, and most preferably to within the normal range of about 4 to about 7 mM.
Because of the relatively large size of the PALP enzyme, its partition into target tissues after administration is expected to be slow. Preferably, PALP is administered several hours prior to an expected glucose load; more preferably PALP is administered at least about 12 hours prior to the expected glucose load; most preferably, PALP is administered at least about 24 hours prior to the expected glucose load.
In another embodiment of the method, purified human PALP, or said active derivative, is administered to a diabetic human. The diabetic human may be afflicted with either Type 1 or Type 2 diabetes.
In the treatment of a diabetic human, administration of purified human PALP may be used in combination with insulin or any anti-diabetic medicament (i.e. biguanides, insulin secretogogues such as sulphonylureas or meglitinides, inhibitors of α-glucosidase, thiazolidinediones, and others). In this embodiment, the anti-diabetic medicament is administered as part of a planned course of treatment for diabetes, in conjunction with purified human PALP. The anti-diabetic medicament may be administered orally or by any other conventional method. While some anti-diabetic medicaments may work in combination with PALP more effectively than others, presently no contra-indication for using any medicament in combination with PALP has been observed
For fine-tuning of glucose level in PALP-treated diabetic patients, a recently developed long-acting derivative of glucagon-like peptide-1 (GLP-1), NN2211, may be especially useful as the anti-diabetic medicament. NN2211 has the useful property that it enhances insulin secretion by the islet only at higher than normal blood glucose levels [Rolin, B., Larsen, M.O., Gotfredsen, C.F., Deacon, C.F., Carr, R.D., Wilken, M. and Knudsen, L.B., "The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases β-cell mass in diabetic mice." Am J. Physiol. Endocrinol. Metab. 283, E745-E752 (2002)].
In the combination of treatments, the patient's situation will determine if the administration of PALP occurs prior to or after administration of the anti-diabetic medicament. For Example, if glycemic control is required very rapidly, then insulin will be administered first followed by administration of PALP; in this embodiment administration of PALP will ensure long-lasting glycemic control.

Treatment Regimen for Treating Diabetes

In another embodiment, the invention provides a treatment regimen for treating diabetes, comprising periodically administering to a diabetic human a therapentically effective amount of purified human placental alkaline phosphatase, or said active derivative. The method may be used in combination with administration of an anti-diabetic medicament. The method maybe effective to maintain the human's blood glucose level below about 10 mM, and preferably in the normal range of 4 mM to 7 mM.
The treatment regimen employs he same active component, purified human placental alkaline phosphatase or said active derivative, described above. The active component may be obtained and purified as described above.
Administration of purified human PALP in the treatment regimen may also be carried out as described above. PALP preparations described herein are also suitable for use in the treatment regimens.
As used with respect to the treatment regimens described herein, the term "periodically" refers to repeated administration of purified human PALP targeted to maintaining blood glucose level within a desired range over the time of treatment. The term "periodically" includes repeated administration at fixed intervals, but also includes repeated administration over irregular intervals as is required by the patient's condition. Furthermore, in the treatment regimens of this embodiment, the therapeutically effective amount of purified human PALP that is administered does not need to be identical for each separate administration. More or less purified human PALP may be administered in separate administrations, as the patient's needs dictate.
As will be appreciated by those skilled in the art, the dosage and the number of treatments will be dependent on the severity of diabetes and the tolerance of the individual patient. Preferably, about 0.2 to about 3 g/m² is administered to the patient once daily; more preferably, about 0.2 to about 1.0 g/m² is administered to the patient once daily; even more preferably, about 0.2 to about 3 g/m² is administered to the patient once or twice weekly; and most preferably about 0.2 to about 3 g/m² is administered to the patient once biweekly.
It is expected that in severe cases of both Type 1 and Type 2 diabetes, once weekly, twice weekly, or daily administration of PALP or active derivative will result in significant reduction of blood glucose level The treatment regimen may be effective to maintain the human's blood glucose level below about 10 mM, preferably below about 8 mM, and most preferably in the normal range of 4 mM to 7 mM.
Furthermore, administration of an anti-diabetic medicament in combination with the PALP may help to fully normalize glucose levels. In the treatment regimens of this embodiment, administration of purified human PALP may be used in combination with insulin or any anti-diabetic medicament (i.e. biguanides, insulin secretogogues such as sulphonylureas or meglitinides, inhibitors of α-glucosidase, thiazolidinediones, and others) to ensure fine-tuning of glycemic control as required. The anti-diabetic medicament may be administered orally or by any other conventional method. A recently developed long-acting derivative of glucagon-like peptide-1 (GLP-1), NN2211, may be especially useful. While some of the anti-diabetic medicaments may work in combination with PALP more effectively than others, presently no contra-indication for using any medicament in combination with PALP has been observed.
By treating a diabetic patient using the treatment regimens described herein, control over diabetes-related conditions may be attained. For example, treatment by the treatment regimens maybe effective to reduce diabetes-associated weight loss for a patient that would normally experience weight loss when treated by an alternative method. Furthermore, the treatment regimens may be effective in treating other diabetes-related complications that have been linked to hyperglycemia. Diabetes-related complications related to hyperglycemia include blindness, renal failure, and various neuropathies [Brownlee, M., "Biochemistry and molecular cell biology of diabetic complications." Nature 414, 813 (2001)]

Method for Inducing Weight Loss or Reducing Weight Gain

Another embodiment of the invention provides a method for treating a human to induce weight loss or to reduce weight gain, comprising regularly administering purified human placental alkaline phosphatase, or said active derivative, to the human in an effective amount to induce weight loss, to reduce an expected weight gain, or to maintain a constant body weight for the human over time.
The method may be particularly suitable for treatment of a human afflicted with Type 2 diabetes mellitus. The method may then be effective to induce weight loss or to reduce an expected weight gain preceding, caused by, or associated with diabetes.
The method may also be suitable to treat an obese person, whose blood glucose level is not elevated, to induce weight loss. Furthermore, the method may be suitable to treat a human who is obese and non-diabetic, but who has a chronically elevated blood glucose level prior to beginning treatment. In this embodiment, the treatment may be effective in preventing the onset of diabetes.
As used herein, the term "obese" refers to a condition where a person's body mass index (BMI) is about 30 kg/m² or greater. Body mass index is defined as a person's mass (in kilograms) divided by the square of the person's height (in meters). Persons who are in the obese category are known to be at greater risk for developing increased blood glucose (or "impaired glucose tolerance"), insulin insensitivity, and eventually Type 2 diabetes.
The method furthermore may be useful to induce weight loss or reduce an expected weight gain preceding the onset of diabetes for a person at risk for Type 2 diabetes. In this embodiment, the treatment maybe effective in preventing the onset of diabetes.
The method employ same active component, purified human placental alkaline phosphatase or said active derivative, described above. The active component may be obtained and purified as described above.
Administration of purified human PALP in the method may also be carried out as described above. By way of example only, the step of administering may be performed by injection of a preparation comprising a physiologically acceptable carrier and human placental alkaline phosphatase or active derivative, dissolved or dispersed in the carrier. The mode of injection maybe intravenous, subcutaneous, intraperitoneal, intramuscular, or intradermal.
PALP preparations described herein are suitable for use in the methods. Byway of example, one suitable preparation comprises homogeneous purified human placental alkaline phosphatase.
As used with respect to the methods described herein, the phrase "regularly administering" refers to repeated administration of purified human PALP targeted to induce weight loss or reduce an expected weight gain over the time of treatment. The expected weight gain may be caused by or associated with the onset of diabetes, for example. The term "regularly" includes repeated administration at fixed intervals, but also includes repeated administration over irregular intervals. Furthermore, in the methods of this embodiment, the effective amount of purified human PALP that is administered does not need to be identical for each separate administration. More or less purified human PALP may be administered in separate administrations, as the patient's needs dictate.
The step of regular administration may include administration of human placental alkaline phosphatase or said active derivative about once per two weeks in some embodiments. In other embodiments, the step of regular administration may include administration of human placental alkaline phosphatase or said active derivative about once per week, about twice per week, or even about once per day.
By way of example only, the effective amount may be in the range of about 0.2 grams to about 3 grams per square meter of calculated surface area for the human. In one embodiment, the effective amount is in the range of about 0.2 grams to about 1 gram per square meter of calculated surface area for the human. In another embodiment, the effective amount is in the range of about 1 gram to about 3 grams per square meter of calculated surface area for the human.

Physiologically Acceptable Preparation of Homogeneous PALP

In another embodiment, the invention provides a preparation for administration to a human, the preparation comprising homogeneous purified human PALP in a physiologically acceptable carrier. Human PALP may be obtained as described above, and maybe purified to homogeneity by the purification procedures described in Example 1. Byway of example, the preparation may be a PALP-containing solution in which homogeneous purified human PALP is dissolved or dispersed in physiological saline. Alternatively, the preparation may comprise homogeneous purified human PALP dissolved or dispersed in another physiologically acceptable carrier, or enclosed in liposomes such as immunoliposomes, or other delivery systems or formulations as are known to the art.
The preparation may suitably contain, for example, about 1 to about 30 mg homogeneous purified human PALP per milliliter of preparation. More suitably, the preparation may contain about 3 to about 15 mg homogeneous purified human PALP per milliliter of preparation. As an example only, one suitable preparation comprises homogeneous purified human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 10 mg/mL. Another suitable PALP preparation comprises homogeneous purified human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 30 mg/mL.

Examples

Example 1. Purification and spectrophotometric assay of PALP.

Human PALP (Type XXIV, 1020 units of total activity) in a partially purified form was obtained commercially from Sigma ChemicaL A butanol extraction of placental tissue, followed by one or more chromatographic steps, was performed by Sigma Chemical to obtain the partially purified material. Butanol extraction inactivates most of the other placental proteins, including growth factors, but does not reduce either the mitogenic or the enzymatic activity of PALP.
As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified PALP obtained from Sigma ("commercial PALP") was not homogeneous and contained other proteins. Fig. 1 shows a picture of a gel separation of a preparation comprising commercial PALP without further purification, and other preparations of PALP of increasing purity. Lane 2 represents a preparation comprising commercial PALP, lanes 3 and 4 represent preparations comprising commercial PALP material after further purification steps (described below), and lane 5 represents a preparation of homogeneous purified PALP obtained by the complete purification procedure described below. Lane 1 contains various molecular mass standards for comparison.
As can be seen by reference to Fig. 1 at lane 2, the preparation comprising commercial PALP contained proteins other than PALP, and did not yield a homogeneous band in the electrophoretic separation. The preparation comprising commercial PALP contains at least three major proteins (one is PALP at approximately 66 kDa, while a band at approximately 52 kDa is α₁-antitrypsin) and several minor proteins. Referring to lane 5 of Fig. 1, the preparation comprising homogeneous purified PALP (obtained by the complete purification procedure described below) apparently contains only PALP.
A purification procedure consisting of several steps was performed to further purify the commercially obtained PALP and to yield a homogeneous band in electrophoretic separation. The same purification procedure was followed that is described elsewhere [She, Q.-B., Mukherjee, J.J., Huang, J.-S., Crilly, K.S. and Kiss, Z., "Growth factor-like effects of placental alkaline phosphatase in human and mouse embryo fibroblasts." FEBS Lett. 469, 163-167 (2000)].
A preparation of partially purified PALP was prepared by dissolving 350 mg of commercial PALP into 10 mL of buffer A (0.1 M sodium acetate, 0.5 M NaCl, 1 mM MgCl₂, 1 mM CaCl₂, adjusted to pH 6.5). This preparation was then further purified by successive Concanavalin A-Sepharose and Q-Sepharose chromatography, essentially following the procedure described elsewhere [Chang, T.-C., Huang, S.-M., Huang, T.-M. and Chang, G.-G., "Human placenta alkaline phosphatase: An improved purification procedure and kinetic studies." Eur. J. Biochem. 209, 241-247 (1992)], as follows.
The preparation was run through a Concanavalin A-Sepharose column using buffer A as solvent. For elution, buffer A included 50 mM α-methyl-D-mannopyranoside. Active fractions collected from the effluent were pooled and dialyzed against buffer B (50 mM Tris-HCl at pH 7.7). SDS-PAGE separation of the collected and dialyzed fraction is shown in Fig. 1 at lane 3.
The collected and dialyzed fraction from the previous step was then passed through a Q-Sepharose column. The fraction of interest was eluted with buffer B using a linear gradient of 0-250 mM potassium phosphate at a pH of 7.5. The active fractions from the Q-Sepharose column were pooled and dialyzed against phosphate-buffered saline and concentrated by Amicon ultrafiltration. SDS-PAGE separation of the collected and dialyzed fraction is shown in Fig. 1 at lane 4, which demonstrates that at least two major proteins are still present in the fraction after dialysis.
Then, the collected and dialyzed fraction from the previous step was purified to homogeneity by t-butyl hydrophobic interaction chromatography (HIC). Prior to adding the fraction to a t-butyl HIC column, the fraction was made 2 M in ammonium sulfate, and pH was adjusted to 6.8. The 5 mL bed volume t-butyl HIC cartridge (BIO-RADIATION, Hercules, California) was connected to a fast performance liquid chromatography (FPLC) system from PHARMACIA (Peapack, New Jersey). The fraction was introduced to the HIC column, and the column was eluted with buffer C (100 mM sodium phosphate buffer, 2 M ammonium sulfate at pH 6.8). The column was eluted with buffer C until a first protein-containing fraction completely eluted, and then a negative gradient of 2 M-0 M ammonium sulfate in 100 mM sodium phosphate at pH 6.8 was passed over the column. The negative linear gradient was used to elute a second protein containing fraction, which contained the enzymatically active PALP protein.
The enzymatically active fraction from the HIC separation was dialyzed against phosphate-buffered saline and concentrated by Amicon ultrafiltration. Presence and purity of the PALP enzyme in the fraction was confirmed by SDS-PAGE. After electrophoretic separation, the gel was stained using coomassie blue or silver stain for visual observation of protein bands. A single protein band was observed with an approximate molecular weight of 66 kDa (Fig. 1, lane 5). Identification of the PALP band by sequence analysis was performed by the Mayo Clinic Protein Core Facility (Rochester, Minnesota).
PALP enzyme activity was assayed spectrophotometrically by monitoring the hydrolysis of 4-nitrophenylphosphate (as an increase in absorbance at 410 nm) at room temperature (22°C) as described elsewhere [Chang, G.-G., Shiao, M.-S., Lee, K.-R. and Wu, J.-J., "Modification of human placental alkaline phosphatase by periodate-oxidized 1,N6-ethenoadenosine monophosphate." Biocham J. 272, 683-690 (1990)]. Activity analysis of 5-10 µg purified enzyme was performed in 1 mL incubation volume containing 50 mM Na₂CO₃/NaHCO₃, 10 mM MgCl₂, 10 mM 4-nitrophenylphosphate at pH 9.8. The extinction coefficient of 4-nitrophenol was taken as 1.62 x 10⁴ M^-1 cm^-1. An enzyme activity of 1 U (unit) is defined as 1 µmol substrate hydrolyzed/min at 22°C at pH 9.8.

Examples 2-8- Effects of PALP on blood glucose levels and body weight in mice.

Example 2. Determination of blood glucose levels in treated and untreated healthy mice in glucose tolerance tests.

Two preparations of commercial PALP, without further purification, were prepared. A first preparation included 30 mg/mL commercial PALP in a 0.9 N physiological salt solution. A second preparation included 10 mg/mL commercial PALP in a 0.9 N physiological salt solution.
First-generation hybrid BDF1 (C57B1/6 female x DBA/2 male) adult (10 to 12 weeks old) male mice, weighing 22-23 g each, were used in the experiment. These animals, developed in the National Cancer Institute (Budapest, Hungary), are in the specified pathogen-free (SPF) hygienic category. The subject animals were kept in macrolon cages at 22-24° C and 50-60% humidity, with lighting regimen of 12 hours light/12 hours dark The animals had free access to tap water and were fed a sterilized standard diet (Charles River VRF1, autoclavable; Germany) according to the provided instructions. The animals were cared for according to "Guiding Principles for the Care and Use of Animals" based upon the Helsinki Declaration.
The mice were fasted for 16 hours before administration of glucose. Four groups of mice were treated and observed in this experiment. A first group was treated by intraperitoneal injection of 500 µg commercial PALP (50 µL injection of the second preparation) exactly 16 hours prior to intraperitoneal administration of glucose (2 mg/mouse). A second group was treated by intraperitoneal injection of 1500 µg commercial PALP (50 µL injection of the first preparation) 24 hours prior to intraperitoneal administration of glucose (2 mg/mouse). A third group was treated by 0.5 I.U. insulin, 15 minutes prior to administration of glucose (2 mg/mouse). A control group received only intraperitoneal administration of glucose (2 mg/mouse).
Blood glucose concentration was monitored for mice of each group, over a period of six hours after administration of glucose. Blood samples were taken from the corner of the eyes (canthus), and glucose concentrations in whole blood samples were immediately measured with the fast Glucose Ctest (Wako Chemicals USA Inc., Richmond, Virginia). Data is shown in the graph of Fig. 2.
The data shown in Fig. 2 demonstrates that, for mice of the control group (●), glucose blood levels increased 3.2 to 4.3-fold within 0.5 to 6 hours after intraperitoneal glucose administration, with a maximum increase at the 3-hour time point. For mice receiving insulin treatment (▲), insulin was effective to initially (i.e., at 30 minutes) block a sharp increase in blood glucose level, but at later time points it was progressively less effective, and at the 6-hour time point it had no significant effect. For mice receiving 500 µg PALP (■), an effect was observed similar to that observed with insulin at the 1-, 3-and 5-hour time points. Finally, for mice receiving 1500 µg PALP (◆), the PALP treatment resulted in greater inhibitory effects on glucose levels than that was observed with insulin at the 1-, 3- and 6-hour time points.
This glucose tolerance experiment clearly indicated that an appropriate amount of PALP may have longer-term effects than insulin when used at a concentration that is safe in animals. In other experiments (not shown), it was determined that for maximal effects PALP needs to be administered about 16 to about 24 hours prior to the glucose load. It is thought that preadministration is more effective because PALP is a relatively large molecule and its distribution in the body takes time. This observation is also an indication that PALP is metabolically stable in vivo. Indeed, others demonstrated that in human subjects injected PALP remains remarkably stable in the circulation with an estimated biological half-life time of 7 days [Clubb, J.S., Neale, F.C. and Posen, S., "The behavior of infused human placental alkaline phosphatase in human subjects." J. Lab. & Clin Med. 66, 493-507 (1965)]. It is expected that, for the treatment regimens described herein, metabolic stability will allow administration of PALP once every 5-14 days, in contrast to practically all anti-diabetic drugs that are presently available, which require daily (often multiple) doses.
Example 3. PALP decreases blood glucose level in streptozotocin-treated BDF1 mice.
BDF1 male mice, weighing 27-28 g each, were used. Four groups of mice were treated and observed in this experiment. A first group and a second group were initially treated by intraperitoneal injection of 1500 µg of commercial PALP (50 µL injection of the first preparation from Example 2). A third group and fourth group were untreated.
Twenty-four hours later (day 0), the first group and third group were treated once with streptozotocin (STZ) at a dosage of 200 mg/kg. STZ is widely used to selectively destroy insulin-producing β-cells in the islet by an oxidative mechanism. The second group and fourth group were untreated at this time. Mice in the first and second groups were then repeatedly administered 1500 µg commercial PALP by intraperitoneal injection (50 µL injection of the first preparation from Example 2) on days 2, 3, 4, and 5.
Blood glucose concentration was monitored for five mice of each group, over a period of six days after administration of STZ (day 0). Blood samples were taken from the eyes, and glucose concentrations in whole blood samples were immediately measured by the Glucose C test. Blood glucose concentration was determined on days 0, 3, 4, 5 and 6.
Data is shown in the graph of Fig. 3
The data shown in Fig. 3 demonstrates that, for mice of the fourth group (●) (untreated), the basal level of blood glucose was stable over the 6-day observation period. For mice of the second group (▲), administration of PALP did not increase, and, importantly, did not decrease the basal blood glucose level. For mice of the third group (■), treated with STZ in the absence of PALP, blood glucose level increased 9-fold by the third day and 10.9-fold by the sixth day. For the first group (◆), treatment with PALP decreased the effect of STZ by 63-67%.
These experiments clearly indicate that repeated administration of PALP is effective in reducing blood glucose levels for mice having damaged β-cells.

Example 4. PALP decreases blood glucose levels even after pre-treatment with STZ.

BDF1 male mice were initially treated with STZ (200 mg/kg). Two weeks after the initial treatments, the mice were treated with 1500 µg commercial PALP (50 µL injection of the first preparation from Example 2) once daily for 5 days, and blood glucose was determined as before on the sixth day. PALP treatment reduced the average blood glucose level from 22.5 mM (n=5) for a control group to 11.5 mM (n=5) for the PALP-treated group.
This experiment strongly indicates that the effect of PALP on blood glucose levels is independent of insulin, because in STZ-treated animals practically no glycemic control remains (as shown by the ∼10-times increase in glucose level compared to the fourth group of Example 3), which is consistent with the massive death of β-cells and the strong reduction or absence of insulin in the circulation. No attempt was made to determine the potentially greater effects of higher concentrations of PALP.

Example 5. PALP decreases the loss of body weight induced by treatment with STZ, and increases life span of STZ-treated animals.

In the same experiment described in Example 3, the weight of each of the mice was measured on days 0, 2, 4, 6, and 8. Data is plotted in Fig. 4, which shows that mice from the untreated fourth group (●) and PALP-treated animals of the second group (▲) gained little, if any, weight during the 8-day observation period. Mice of the third group (■) treated with STZ in the absence of PALP lost approximately 5 grams. Weight loss for mice of the first group (◆) treated with both PALP and STZ was inhibited about 50%.
It is known that in the absence of insulin, protein degradation in muscles is enhanced leading to weight loss (wasting). In STZ-treated animals, this mechanism almost certainly plays a key role in weight loss. In unpublished work, Kiss and co-workers found that in differentiated L6 muscle cells, administration of PALP increases protein synthesis from various amino acids (alanine, proline, leucine, glutamine, methionine). Thus, stimulation of protein synthesis in muscle is likely to explain, at least in part, the ability of PALP to partially prevent weight loss in STZ-treated animals.
PALP, administered either subcutaneously or intraperitoneally, had a profound effect on the survival of STZ-treated animals. Of the 15 animals treated with STZ alone, only 6 survived up to 6 months. On the other hand, all 23 animals treated with STZ (one time) plus 1.5 mg PALP/mouse (for at least five consecutive days) survived for at least 8 months. Because conventionally a high survival rate of STZ-treated animals can be ensured only with daily injection of insulin, the positive effects of PALP on animal survival further indicate that PALP has insulin-like, but insulin-independent, physiological effects, including reduction of blood glucose level. Reduction in blood glucose level and increased protein synthesis together may explain the ability of PALP to promote survival of these animals.

Example 6. Determination of blood glucose levels in untreated and PALP-treated non-obese diabetic mice.

In this set of experiments, the effect of PALP on blood glucose levels was determined in non-obese diabetic adult female mice. Data for the experiments is given in Table 2.
Non-obese (NOD) diabetic inbred adult (140-150 days old) female mice weighing 22-26 g each were obtained from Charles River Italia S.P.A. (Italy). The NOD mouse strain is a spontaneous animal model of human Type 1 diabetes isolated from the cataract-prone CTS subline of outbred ICR mouse. The mice are also in the specified pathogen-free (SPF) category. These mice are characterized by a cumulative incidence of diabetes, reaching 60-80% in females and 10-20% in males at an age of 150-200 days. In these mice, spontaneously autoreactive T-cells destroy the insulin-producing islet β-cells in the pancreas.
The following references provide a summary of the current understanding on how diabetes develops in NOD mice: Lyons, P.A., Armitage, N., Lord, C.J., Phillips, M.S., Todd, J.A., Peterson, L.B. and Wicker, L.S., "Mapping by Genetic Interaction: High resolution congenic mapping of the ); Kaufman, D.L., Tisch, R, Sarvetnick, N., Chatenoud, L., Harrison, L.C., Haskins, K., Quinn, A., Sercarz, E., Herrath, M., Wegmann, D., Wen, L. and Zekzer, D., "Report from the 1st international NOD mouse T-cell workshop and the follow-up mini-workshop." ); Bonifacio, E., Atkinson, M., Eisenbarth, G., Serreze, D., Kay, T.W.H., Lee-Chan, E. and Singh, B., "International Workshop on lessons from animal models for ).
The subject animals were kept in macrolon cages at 22-24° C and 50-60% humidity, with lighting regimen of 12 hours light/12 hours dark The animals had free access to tap water and were fed a sterilized standard diet (Charles River VRF1, autoclavable; Germany) according to the provided instructions. The animals were cared for according to "Guiding Principles for the Care and Use of Animals" based upon the Helsinki Declaration.
For treated and control NOD animals (180 days old), blood samples were taken from the eye corner, and initial blood glucose levels (day 1) were determined by the Fast Glucose C test. Over an eleven-day period, a solution of commercial PALP (30 mg/mL in physiological saline) was injected into four treated animals (1.5 mg/mouse) subcutaneously (s.c.) or intraperitonially (i.p.) at four times. Injections were given on days 1, 3, 5 and 8. On day 11, blood samples were taken from the eye corner for both treated and control animals, and blood glucose levels were determined by the Fast Glucose C test.

As indicated by the data in Table 2, for the three untreated NOD mice, blood glucose concentrations increased by 7.2-8.4 mM during the 11-day period. In contrast, in the four animals treated with PALP subcutaneously, the increases in blood glucose levels were considerably lower (in the range of 0.6-3.1 mM). For animals treated with PALP intraperitonially, there was a similar reduction in the increase (in the range of 0.7-3.4 mM) in blood glucose levels (data not given in Table 2).

Table 2. Changes in blood glucose levels observed for untreated and PALP-treated NOD mice.

	Untreated Mice			PALP-Treated Mice (subcutaneous)
Subject	1	2	3	1	2	3	4
	Blood glucose level (mM)			Blood glucose level (mM)
Day 1	12.7	8.2	12.9	7.9	12.9	8.1	13.3
Day 11	20.8	15.4	21.3	8.5	16.0	8.7	16.2
Increase	8.1	7.2	8.4	0.6	3.1	0.6	2.9
Survival (days)	12	22	16	<150	25	<150	27

Also, it was observed that the untreated mice survived for not longer than 22 days; in contrast, PALP-treated mice survived for at least 25 days, and up to over 150 days in some cases.
In this set of experiments, no attempt was made to examine the potentially stronger effects of administering a greater quantity of PALP. The results clearly indicate that administration of PALP markedly reduces the rise in blood glucose levels in the non-obese Type 1 diabetic model.

Example 7. Determination of blood glucose levels in untreated and PALP-treated obese diabetic mice.

In this set of experiments, the effect of PALP on blood glucose levels was determined in obese diabetic adult female mice. Data for the experiments is given in Table 3.
Obese diabetic inbred adult (six weeks old) female C57BI/6J Bom-ob mice weighing 33-37 g were obtained from Charles River Laboratories, Germany. These animals are in the specified pathogen-free (SPF) hygienic category. A mutation in the leptin gene, responsible for obesity, was propagated in the C57B1/6J (B1/6) inbred strain. These homozygous obese (ob/ob) animals developed hyperglycemia, hyperinsulinemia and obesity by six weeks of age. Food intake is greatly increased by that time.
The subject animals were kept in macrolon cages at 22-24° C and 50-60% humidity, with lighting regimen of 12 hours light/12 hours dark The animals had free access to tap water and were fed with a sterilized standard diet (Charles River VRF1, autoclavable) according to the prescribed standard. The animals were cared for according to "Guiding Principles for the Care and Use of Animals" as stated in the Helsinki Declaration.
On day 43 (Day 1 in Table 3), the animals were divided into five groups (I-V) each consisting of four animals, and the treatments started as specified in Table 3. Animals with similarly high blood glucose levels were selected for each of groups I-V. In short, the treatments were: untreated (Group I); treated with commercial PALP for 28 days, 5 times/week, intraperitonially (Group II); treated with commercial PALP for 28 days, 3 times/week, intraperitonially (Group III); treated with commercial PALP for 28 days, 5 times/week, subcutaneously (Group IV); treated with commercial PALP for 28 days, 3 times/week, subcutaneously (Group V). Initially, each treatment consisted of 1.5 mg commercial PALP/mouse in physiological saline; then on day 15 (Group II and IV) or day 16 (Group II and V) the dosage of commercial PALP was increased to 3 mg/mouse. Blood samples were analyzed for glucose on the days indicated in the table. Blood glucose level (BGL) data are the mean ± S.E. of single simultaneous determinations from four animals of the group.
Data is tabulated in Table 3. For the untreated animals of Group I, blood glucose levels increased steadily over the 28 days period. However, for both intraperitoneal (Group II and III) and subcutaneous administration (Groups IV and V) of PALP, a steady decrease in blood glucose was observed for the subject animals. The data further indicate that the 1.5 mg PALP/mouse dosage was effective in decreasing the blood glucose level, while the 3 mg/mouse dosage was slightly more effective.

It should be noted that intraperitoneal and subcutaneous treatments were similarly effective, with intraperitoneal administration providing somewhat better results. It is also noteworthy that treatment 5 times/week was only slightly more effective than treatment 3 times/week for either intraperitoneal or subcutaneous administration. The data suggests that, in humans, daily administration may not be required in order to effectively control hyperglycemia.

Table 3. Changes in blood glucose levels observed for untreated and PALP-treated obese diabetic mice. (BGL=Blood Glucose Level (mM))

	Group I	Group II intraperitoneal		Group III intraperitoneal		Group IV subcutaneous		Group V subcutaneous
Day	BGL ± S.E.	BGL ± S.E.	PALP	BGL ± S.E.	PALP	BGL ± S.E.	PALP	BGL ± S.E.	PALP
1	5.6	7.2	1.5 mg	7.6		7.1	1.5 mg	7.3
1	± 0.7	± 0.2	1.5 mg	± 0.9		±0.5	1.5 mg	± 0.5
2	-	7.3	1.5 mg	7.6	1.5 mg	7.1	1.5 mg	7.3	1.5 mg
2	-	± 0.2	1.5 mg	± 0.8	1.5 mg	± 0.4	1.5 mg	± 0.6	1.5 mg
3	5.8	6.6	1.5 mg	7.1		6.7	1.5 mg	7.3
3	± 0.9	±0.2	1.5 mg	± 0.6		± 0.2	1.5 mg	± 0.6
4	-	6.4	1.5 mg	6.8	1.5 mg	6.8	1.5 mg	6.8	1.5 mg
4	-	± 0.3	1.5 mg	± 0.2	1.5 mg	± 0.1	1.5 mg	± 0.4	1.5 mg
5	6.2	5.4	1.5 mg	6.4		6.5	1.5 mg	6.4
5	± 0.6	± 0.3	1.5 mg	± 0.5		± 0.1	1.5 mg	± 0.3
7	-	5.1	1.5 mg	6.1	1.5 mg	6.3	1.5 mg	6.2	1.5 mg
7	-	± 0.2	1.5 mg	± 0.7	1.5 mg	± 0.2	1.5 mg	± 0.5	1.5 mg
8	7.6	5.0	1.5 mg	5.5		6.1	1.5 mg	5.8
8	± 1.2	± 0.5	1.5 mg	± 0.7		± 0.1	1.5 mg	± 0.3
9	-	5.2	1.5 mg	5.7	1.5 mg	6.1	1.5 mg	5.7	1.5 mg
9	-	± 0.2	1.5 mg	± 0.6	1.5 mg	± 0.2	1.5 mg	± 0.5	1.5 mg
10	7.9	4.5	1.5 mg	5.5		6.1	1.5 mg	5.5
10	± 0.2	± 0.3	1.5 mg	± 0.6		± 0.3	1.5 mg	± 0.6
11	-	4.4	1.5 mg	5.2	1.5 mg	5.9	1.5 mg	5.3	1.5 mg
11	-	± 0.3	1.5 mg	± 0.4	1.5 mg	± 0.3	1.5 mg	± 0.4	1.5 mg
14	8.6	4.6	1.5 mg	5.1	1.5 mg	5.7	1.5 mg	5.3	1.5 mg
14	± 1.3	± 0.2	1.5 mg	± 0.5	1.5 mg	± 0.3	1.5 mg	± 0.4	1.5 mg
15	-	4.4	3 mg	4.8		5.2 ±0.3	3 mg	5.0
15	-	± 0.1	3 mg	± 0.4			3 mg	± 0.4
16	8.8	3.5	3 mg	4.4	3 mg	4.1	3 mg	4.2	3 mg
16	± 0.4	± 0.3	3 mg	± 0.4	3 mg	± 0.2 3 mg	3 mg	± 0.2	3 mg
17	-	2.9	3 mg	3.4		3.7	3 mg	3.6
17		± 0.2	3 mg	± 0.2		± 0.2	3 mg	± 0.2
18	8.0	2.7	3 mg	3.1	3 mg	3.5	3 mg	3.4	3 mg
18	± 0.2	± 0.2	3 mg	± 0.2	3 mg	± 0.1	3 mg	± 0.3	3 mg
21	-	2.5	3 mg	2.9	3 mg	3.4	3 mg	3.3	3 mg
21		± 0.2	3 mg	± 0.3	3 mg	± 0.3	3 mg	± 0.2	3 mg
22	8.6	2.3	3 mg	2.7		3.6	3 mg	3.0
22	± 0.5	± 0.2	3 mg	± 0.2		± 0.2	3 mg	± 0.4
23	-	2.4	3 mg	2.5	3 mg	3.1	3 mg	2.6	3 mg
23		± 0.3	3 mg	± 0.1	3 mg	± 0.1	3 mg	± 0.3	3 mg
24	8.8	2.3	3 mg	2.2		2.9	3 mg	2.6
24	±0.5	± 0.1	3 mg	± 0.2		± 0.2	3 mg	± 0.3
25	-	2.0	3 mg	2.1	3 mg	2.8	3mg	2.5	3 mg
25		± 0.2	3 mg	± 0.3	3 mg	± 0.2	3mg	signe en epic	3 mg
28	8.5	1.5		1.6		2.3		2.3
28	± 0.4	± 0.1		± 0.1		± 0.3		± 0.2

Example 8. Effect of PALP treatment on body weight for obese diabetic mice.

Obesity is characterized by an increase in adipose tissue and body weight to a level that produces adverse health effects, including increased risk for development of diabetes. White adipose tissue secretes several factors that regulate energy balance and whole-body homeostasis; one of the most important such factors is the hormone leptin [F.M. Gregoire, "Adipocyte differentiation: From fibroblast to endocrine cell." Exp. Biol. Med. 226, 997-2001 (2001)].
The gene responsible for leptin production is called the ob gene. In ob/ob animals, the leptin secreted from the adipose tissue is truncated and thereby inactivated [B.M. Spiegelman and J.S. Flier, "Obesity and the regulation of energy balance." Cell 104, 531-543 (2001)]. Due to the absence of leptin-mediated regulation, ob/ob animals gain substantial weight, leading gradually and inevitably to decreased insulin sensitivity in the peripheral tissues such as muscle, adipose tissue, and liver. As a consequence, in ob/ob animals the blood glucose level is high due to impaired uptake and metabolism of glucose by the peripheral tissues, and to increased production of glucose by the liver, which is normally inhibited by insulin. The obesity syndrome in ob/ob mice can be corrected by administration of leptin [B.M. Spiegelman and J.S. Flier, "Obesity and the regulation of energy balance." Cell 104, 531-543 (2001)].
Importantly, high glucose levels that are induced by physiological events other than obesity do not lead to obesity. Also, lowering of glucose level does not necessarily lead to weight loss; i.e., high glucose level is not a risk factor for obesity. For example, some of the most important agents that are used to treat diabetes by lowering blood glucose level, including insulin, sulphonylureas and thiazolidinediones, can cause significant weight gain [see Table 1 in D.E. Moller, "New drug targets for )], while biguanides (e.g., metformin) can cause moderate weight gain [U.K. Prospective Diabetes Study Group, "U.K )]. None of the presently used glucose-lowering agents are known to cause weight loss.

In view of the differential regulation of adipose tissue weight and blood glucose level, it was of interest to determine whether administration of PALP also modifies body weight that primarily results from increased weight of adipose tissue in ob/ob mice. Accordingly, in the experiment described in Example 7, the body weight of untreated and PALP-treated obese diabetic mice was also regularly measured The schedule of treatments and the results are given in Table 4.

Table 4. Effect of administration of PALP on body weight of obese diabetic mice.

	Group I	Group II intraperitoneal		Group III intraperitoneal		Group IV subcutaneous		Group V subcutaneous
Day	Body weight (g) ± (g) ± S.E.	Body weight S.E.	PALP	Body weight (g) ± S.E.	PALP	Body weight (g) ± S.E.	PALP	Body weight (g) ± S.E.	PALP
1	35.5	37.2	1.5 mg	33.3		33.5	1.5 mg	33.0
1	±0.9	±1.3	1.5 mg	±1.7		±0.6	1.5 mg	±0.7
2	34.4	36.7	1.5 mg	31.7	1.5mg	32.1	1.5 mg	31.6	1.5 mg
2	±1.0	±0.6	1.5 mg	±1.6	1.5mg	±1.0	1.5 mg	±1.1	1.5 mg
3	35.5	36.3	1.5mg	32.2		31.7	1.5 mg	32.7
3	± 0.8	± 0.5	1.5mg	± 1.4		± 1.0	1.5 mg	±0.8
4	35.6	35.8	1.5mg	31.7	1.5 mg	31.5	1.5 mg	31.6	1.5 mg
4	±0.9	±0.7	1.5mg	±1.9	1.5 mg	±0.9	1.5 mg	±0.5	1.5 mg
5	36.0	35.3	1.5mg	31.8		32.1	1.5 mg	31.7
5	±1.1	±0.7	1.5mg	±1.5		±1.2	1.5 mg	± 0.5
7	37.3	-	1.5 mg	32.3	1.5 mg	32.9	1.5 mg	33.2	1.5 mg
7	±0.9		1.5 mg	±1.3	1.5 mg	±0.9	1.5 mg	±0.3	1.5 mg
8	38.5	35.9	1.5 mg	32.2		33.4	1.5 mg	32.4
8	±1.3	±0.6	1.5 mg	±2.1		±0.6	1.5 mg	±1.1
9	38.0	35.4	1.5 mg	32.2	1.5 mg	33.7	1.5 mg	32.8	1.5 mg
9	±1.8	±0.8	1.5 mg	±1.6	1.5 mg	±0.9	1.5 mg	±0.6	1.5 mg
10	37.9	35.3	1.5 mg	32.3		33.7	1.5mg	32.2
10	± 1.0	±0.5	1.5 mg	±1.6		±1.6	1.5mg	±0.5
11	37.9	35.0	1.5mg	32.3	1.5 mg	33.8	1.5 mg	32.9	1.5 mg
11	±1.3	±0.4	1.5mg	±1.9	1.5 mg	±1.9	1.5 mg	±0.4	1.5 mg
14	39.7	33.9	1.5 mg	33.0	1.5 mg	34.8	1.5 mg	34.2	1.5 mg
14	±1.2	±1.0	1.5 mg	±1.2	1.5 mg	±2.3	1.5 mg	±1.0	1.5 mg
15	39.7	34.0	3 mg	33.0		36.0	3 mg	33.7
15	±1.0	±1.9	3 mg	±1.9		±1.0	3 mg	±0.5
16	39.9	33.8	3 mg	32.9	3 mg	34.7	3 mg	33.9	3 mg
16	±1.4	±0.8	3 mg	±2.1	3 mg	±2.1	3 mg	±0.5	3 mg
17	39.8	33.5	3 mg	32.6		34.8	3 mg	33.8
17	±1.5	±1.1	3 mg	±2.2		±2.2	3 mg	±0.4
18	40.1	33.3	3 mg	32.8	3 mg	35.2	3 mg	34.1	3 mg
18	±1.3	±1.0	3 mg	±2.3	3 mg	±1.6	3 mg	±0.4	3 mg
21	41.4	33.6	3 mg	33.7	3 mg	36.2	3 mg	36.2	3 mg
21	±1.7	±1.2	3 mg	±2.4	3 mg	±1.7	3 mg	±0.7	3 mg
22	41.7	33.1	3 mg	33.5		35.9	3 mg	36.0
22	±1.6	±1.8	3 mg	±1.8		±1.7	3 mg	±1.0
23	41.6	-	3 mg	33.4	3 mg	35.6	3 mg	--	3 mg
23	±2.8		3 mg	±2.3	3 mg	±1.7	3 mg		3 mg
24	41.8	-	3 mg	33.2		35.8	3 mg	-
24	±2.0		3 mg	+1.5		±1.6	3 mg
25	41.6	32.2	3 mg	32.6	3 mg	36.1	3 mg	37.1	3 mg
25	±1.4	±1.3	3 mg	±1.4	3 mg	±2.1	3 mg	±0.8	3 mg
28	43.7	32.8		32.9		37.4		37.4
28	±2.7	±0.8		±1.4		±1.6		±1.0
% Gain	23.1%	-11.8%		-1.2%		11.6%		13.3%

Untreated animals of Group I steadily gained weight over the 28-day observation period, with body weight increasing approximately 23% overall. In contrast, animals treated with PALP administered intraperitoneally five times per week (Group II) actually decreased in weight by approximately 12% overall, while animals treated with PALP administered intraperitoneally three times per week essentially maintained their weight (Group III). Animals treated with PALP five times per week (Group IV) administered subcutaneously, or three times per week (Group V) administered subcutaneously gained about half as much weight as the control group.
Because intraperitoneal and subcutaneous treatments had similar effects on the blood glucose level (Table 3), the clearly established ability to achieve weight control by intraperitoneally administering PALP may involve some mechanism in addition to the control of glucose level. In fact, considering that for the ob/ob model obesity leads to insulin insensitivity and higher blood glucose levels, but not vice-versa, it is likely that in this model a primary mode of action of PALL was weight control, and that the reduction in blood glucose level was a secondary effect. The mechanism by which PALP achieves weight control is not known. However, since the absence of functional leptin hormone is responsible for obesity in ob/ob animals, it is reasonable to assume that PALP acts as a substitute for the most important weight control related functions of leptin.

Examples 9-12 - Effects of PALP on glucose uptake and metabolism in differentiated 3T3-L1 adipocytes and rat L6 cells at different levels of glucose and serum in the medium.

The above-described experiments suggested that PALP decreased the blood glucose level in treated mice by stimulating glucose uptake and metabolism in peripheral tissues, which explains the relatively long time required for body distribution and action. This hypothesis was examined for differentiated adipocytes and muscle cells.
Mouse 3T3-L1 pre-adipocyce cells and rat L6 skeletal muscle cells were obtained from the American Type Culture Collection (Rockville, Maryland). 3T3-L1 cells were grown in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic/antimycotic (GIBCO, Grand Island, New York) composed of 10,000 units/mL penicillin G, 10 mg/mL streptomycin, and 25 µg/mL amphotericin B.
To induce cell differentiation of the two cell lines, conventional methods were used, as follows. 3T3-L1 cells were maintained in a state of confluency for 2 days, followed by treatments for 3 days with 400 nM insulin (Boehxinger Mannheim, Indianapolis, Indiana), 250 nM dexamethasone (Sigma-Aldrich, Inc., St. Louis, Missouri) and 0.5 mM isobutyimethylxanthine (Sigma-Aldrich, Inc.). The medium was replaced with fresh 10% serum-containing medium, and the medium was changed twice a week. The differentiated cells were used 14 days after removing the differentiation-inducing agents.
The L6 cells in monolayer culture were induced to differentiate in minimal essential medium (MEM) containing 2% (v/v) FBS and 1% (v/v) antibiotic/antimycotic solution. The cells were fed fresh medium every 48 hours. Myoblast differentiation, which occurred by about the seventh day, was monitored by phase contrast microscopy.
To examine cellular glucose uptake and metabolism, differentiated (attached) cells in 12-well plates were incubated with D-[¹⁴C]glucose at 37°C in a CO₂ incubator (95% air. 5% CO₂) in 2% serum- or 10% serum-containing (as specified in the specific examples) glucose-free or 5 mM D-glucose-containing medium for 2-6 hours. In the various experiments, the quantityof other reagents (such as PALP- or insulin) in the incubation medium was varied, as specified.
At the termination of incubation, the medium was removed; an aliquot of the medium was taken to determine the loss of D-[¹⁴C]glucose from the medium, which corresponds to glucose uptake by cells. Cells were washed twice with 3-3 mL medium to remove traces of medium containing unincorporated radioactivity. Then ice-cold 99.8% methanol/0.2% water (v/v) mixture (1 ml) was added to the monolayers, and cells were extracted for 2 hours at -20° C This treatment resulted in the precipitation of glycogen and the solubilization of cellular free glucose and lipids.
Precipitated [¹⁴C]glycogen was suspended in 0.75 mL of 1 M NaOH and transferred to scintillation vials; this procedure was repeated with another 0.75 mL aliquot of NaOH 10 mM HCl (∼150 µL) was added to the vials to neutralize the suspension, followed by the addition of 8.5 mL ECOLUME (ICN Pharmaceuticals, Inc., Costa Mesa, California). While the 1 M NaOH suspension contained some particulate material (mostly protein which was not labeled with radiolabeled glucose), after transfer to ECOLUME no precipitate remained. This procedure resulted in quantitative removal of precipitated glycogen; no additional radioactivity could be removed from the well by 30% KOH (which was neutralized with HCl prior to determination of radioactivity.
The methanol mixture containing cellular free glucose and lipids was treated as follows. The methanol phase (1 ml) and a following 1 mL methanol wash were added to 2 mL chloroform, followed by the addition of 3 mL water. Separation of this organic-aqueous mixture was facilitated by brief centrifugation. Of the resulting two phases, the lower one contained the total radiolabeled lipids, while the upper one contained radiolabeled glucose. Aliquots of the upper and lower phases were transferred to scintillation vials to determine the amounts of radiolabeled glucose and total lipids, respectively, by liquid scintillation counting.
In some experiments, a published procedure [Huang, D., Cheung, AT., Parsons, J.T. and Bryer-Ash, M., "Focal adhesion kinase (FAK) regulates insulin stimulated glycogen synthesis in hepatocytes." J. Biol. Chem. 277, 18151-18160 (2002)] parallel to the above method was used to determine glucose incorporation into glycogen. After incubations with D-[¹⁴C]glucose, cells were solubilized with 20% KOH for 2 hours. Lysates were extracted with 8% tricarboxylic acid, neutralized with 2.0 M HCl, then boiled for 5 minutes. Total glycogen was precipitated by the addition of 80% ethanol (final concentration) for 2 hours at -20° C followed by centrifugation at 1100 x g for 10 minutes. Precipitation and centrifugation was repeated. The precipitate was then redissolved in distilled water, and the solids were precipitated again as before. This method yielded essentially the same results as the methanol precipitation method described above. Thus, in all subsequent experiments we used the methanol precipitation method because this permitted simultaneous analysis of cellular glucose, glycogen and lipids for an individual sample.

Example 9. Stimulatory effect of PALP on glucose uptake in differentiated L1 cells.

Three groups of differentiated L1 cells (used 14 days after the differentiation treatment described above) in 12-well plates were treated as follows: a first control group was untreated; a second group was treated with 0.75 mL of a preparation comprising 0.5 U/mL commercial PALP in serum-free and glucose-free DMEM for 30 minutes, and a third group was treated with 0.75 mL of a preparation comprising 1.0 U/mL commercial PALP in serum free and glucose-free DMEM for 30 minutes. The following experiment was performed in triplicate for each group.
Each group was then incubated as described above for 5 hours following addition of D-[¹⁴C]glucose, under four different conditions as follows: (1) the incubation medium contained no serum or unlabeled D-glucose; (2) the incubation medium contained 2% fetal bovine serum (FBS) but no unlabeled D-glucose; (3) the incubation medium contained 5 mM unlabeled D-glucose but no serum; and (4) the incubation medium contained 5 mM unlabeled D-glucose + 2% FBS.
For each condition for each group, the amount of radiolabeled glucose was determined by liquid scintillation counting. Results are charted in Fig. 5. The data are the mean ± S.D. of three determinations for each group under each condition. Under each of the four conditions, treatment with PALP at both 0.5 U/mL
and 1.0 U/mL (■) concentrations enhanced the cellular content of D-[¹⁴C]glucose approximately two-fold or more, relative to the untreated control group (□). In the presence of 5 mM unlabeled D-glucose (conditions 3 and 4), L1 cells obviously incorporated less D-[¹⁴C]glucose (because of greatly decreased specific activity); at the same time PALP had somewhat greater stimulatory effects on D-[¹⁴C]glucose uptake compared to its effect in glucose-free medium. The experiment was repeated for each group at least once, with similar results.

Example 10. Stimulatory effect of PALP on cellular synthesis of [¹⁴C]glycogen from D-[¹⁴C]glucose.

The same conditions as described in Example 9 were used, except the amount of radiolabeled glycogen was determined by liquid scintillation counting. Results are charted in Fig. 6. Treatments included PALP at 0.5 U/mL
and 1.0 U/mL (■), and the untreated control group (□). Again, while in the presence of 5 mM unlabeled D-glucose L1 cells obviously incorporated less D-[¹⁴C]glucose into glycogen, in the presence of 5 mM D-glucose PALP had slightly greater (∼2 to ∼3-fold) stimulatory effects on the synthesis of [¹⁴C]glycogen.

Example 11. Stimulatory effect of PALP on the incorporation of D-[¹⁴C]glucose into the total lipid fraction.

The same conditions as described in Example 9 were used, except the amount of total radiolabeled lipids was determined by liquid scintillation counting. Results are charted in Fig. 7. Treatments included PALP at 0.5 U/mL
and 1.0 U/mL (■), and the untreated control group (□). Although in the presence of 5 mM unlabeled D-glucose cells incorporated less D-[¹⁴C]glucose into lipids, as expected, PALP had slightly greater (∼2 to ∼2.5-fold) stimulatory effects on [¹⁴C]lipid synthesis compared to its effects in the absence of D-glucose.
Overall the experiments described in Examples 9-11 indicate that, in differentiated L1 cells, PALP clearly stimulates cellular uptake and metabolism of D-glucose, that PALP is more effective at 1 U/mL than at 0.5 U/mL concentration, and that the effects of PALP do not strongly depend on the concentration of glucose in the medium These experiments strongly suggest that a primary target of PALP in mice, and by extension in humans, is fat tissue.

Example 12. Stimulatory effect of PALP on glucose metabolism in differentiated L6 cells.

Another potential target of PALP may be the skeletal muscle. The effects of PALP on glucose uptake/metabolism were determined in differentiated L6 cells, a widely used model for skeletal muscle.
Differentiated L6 cells in 12-well plates were incubated in 2% FBS-containing MEM for 6 hours with 1 µCi/mL of D-[¹⁴C]glucose in the absence (□) or presence of 1 U/mL commercial PALP
, 50 nM insulin ("Ins.)
, or 1 U/mL commercial PALP + 50 nM Ins (■). The data, shown in Fig. 8, are the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results).
PALP clearly stimulated, although only slightly, both the cellular uptake of D-[¹⁴C]glucose and its metabolism to [¹⁴C]glycogen and [¹⁴C]total lipid for the differentiated L6 cells. Insulin was at least 50% more effective than PALP in stimulating all three mechanisms, and in each case the effects of PALP plus insulin were roughly additive or less than additive.

Examples 13 and 14- Digestion with protease does not reduce the effects of PALP on glucose metabolism.

Example 13. Digestion of PALP by bromelain.

Bromelain (BRL) is a protease which was previously shown to effectively digest PALP leading to the formation of fragments of lower molecular mass [Kottel, R.H and Hanford, W.C, "Differential release of membrane-bound alkaline phosphatase isoenzymes from tumor cells by bromelain." Biobem Biopbys. )]. Based on this observation, bromelain was used to digest commercial PALP to determine if digestion by a protease can generate a smaller PALP fragment which is able to stimulate glucose metabolism.
A preparation comprising 20 U/mL commercial PALP and 0.01 mg/mL of BRL (Sigma-Aldrich) in 1 mL of 25 mM Tris-HCl (pH 7.4) buffer was incubated at 37°C for 2 hours. Fig. 9 shows a gel picture (obtained by SDS-PAGE using 7.5% polyacrylamide) with undigested commercial PALP in lane 1 and BRL-digested PALP in lane 2. It is clear that, after digestion with 0.01 mg/mL of BRL, no detectable amount of the original PALP molecule remains.

Example 14. Effect of bromelain digestion product of PALP on glucose metabolism.

Differentiated LI cells were preincubated in serum-free and glucose-free DMEM for two hours under the following conditions: (a) untreated; (b) in the presence of 1 U/mL commercial PALP; (c) in the presence of 0.01 mg/mL of BRL; and (d) in the presence of 1 U/mL commercial PALP + 0.01 mg/mL BRL. Each group was then incubated for 5 hours in the presence of D-[¹⁴C]glucose. Total radiolabeled lipids, radiolabeled glucose, and radiolabeled glycogen were determined by liquid scintillation counting, as described above.
Data is shown in Fig. 10. Treatment by BRL alone
slightly enhanced both [¹⁴C]glycogen and [¹⁴C]lipid synthesis but had no clear effect on the cellular content of D-[^l4C]glucose, relative to untreated cells (□). Treatment with the bromelain digestion product of PALP (■) enhanced glycogen synthesis, lipid synthesis, and glucose uptake relative to untreated cells (□). Treatment with the bromelain digestion product of PALP(■) slightly enhanced glycogen synthesis but not lipid synthesis, and had no clear effect on the cellular content of D-[¹⁴C]glucose, relative to treatment by PALP
.
This experiment indicated that at least one PALP fragment (i.e. a sequence of PALP which is smaller than the full size PALP) at least partially retained the ability of native PALP to enhance glucose metabolism Furthermore, the experiment also justifies the assumption that a fragment of PALP may be even more active in stimulating glycogen synthesis than native PALS. Potentially any sequence derived from PALP could be active in promoting glucose metabolism

Examples 15-17 - Comparison of the effects of PALP and insulin on glucose metabolism in differentiated L1 cells.

During the course of the experiments, it was observed that L1 cells respond better to PALP if the cells are used 8 to 11 days after terminating treatment with differentiation-inducing agents; as opposed to 14 days which was practiced in the previous experiments. Therefore, in the subsequent experiments we used L1 cells within 9 to 11 days after terminating treatment with differentiation-inducing agents.

Example 15. Effect of PALP and Ins on glucose content of incubation medium in differentiated L1 cells.

In this experiment, differentiated L1 cells in 12-well plates were incubated (in 0.75 mL volume/well) in 2% BFS-containing glucose-free DMEM for 6 hours with 1 µCi/mL of D-[¹⁴C]glucose in the absence or presence of 1 U/mL commercial PALP or 50 nM Ins. At the conclusion of the experiment, 0.5 mL aliquots of the incubation medium were added to 9.5 mL ECOLUME, and the radioactivity was determined by scintillation counting. The data indicating loss of D-[¹⁴C]glucose from the medium are the means ± S.E.M. of three experiments each performed in triplicate.
Data is graphed in Fig. 11. The experiment showed that treatment with PALP
and 50 nM Ins (■) stimulated the loss of D-[¹⁴C]glucose from the medium (i.e. uptake by the cells) by ∼86% and 77%, respectively, relative to the untreated control (□). Thus, Ins, at the physiologically supra-maximal 50 nM concentration, was more effective than PALP in increasing the uptake of D-[¹⁴C]glucose by differentiated L1 cells.

Example 16. Effect of PALP and Ins on the cellular uptake and metabolism of glucose in differentiated L1 cells.

Differentiated L1 cells in 12-well plates were incubated in 2% FBS-containing glucose-free DMEM with 1 µCi/mL of D-[¹⁴C]glucose for 2 hours (Fig. 12, upper panel) or 6 hours (Fig. 12, lower panel) in the absence (□) or presence of 0.25 U/mL commercial PALP
,1 U/mL commercial PALP
, or 10 nM Ins (■). The data shown in Fig. 12 are the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results). Note that on this occasion, the concentration of Ins was only 10 nM, which is closer (although still higher) to the physiological concentration of Ins (around 1 nM or less).
After treatments for 2 hours, 1 U/mL PALP had somewhat greater effects than Ins on the cellular level of D-[¹⁴C]glucose and [¹⁴C]glycogen (upper panel. After incubations for 6 hours, 1 U/mL of PALP was more effective than 10 nM Ins in stimulating uptake of D-[¹⁴C]glucose and its subsequent conversion into both [¹⁴C]glycogen and [¹⁴C]total lipid (lower panel).

Example 17. Comparison of the effects of PALP and a high concentration of Ins on the cellular uptake and metabolism of glucose in differentiated L1 cells.

Differentiated L1 cells in 12-well plates were incubated in 2% FBS-containing glucose-free DMEM for 2 hours (Fig. 13, upper panel) or 6 hours (Fig. 13, lower panel) with 1 µCi/mL of D-[¹⁴C]glucose in the absence (□) or presence of 0.25 U/mL commercial PALP
, 1 U/mL commercial PALP
, or 50 nM Ins (■). The data shown in Fig. 13 are the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results).
After incubations for either 2 hours (Fig.13, upper panel) or 6 hours (Fig. 13, lower panel), 50 nM Ins was significantly more effective than 1 U/mL of PALP- on [¹⁴C]glycogen synthesis and slightly more effective on [¹⁴C]total lipid synthesis and the cellular content of D-[¹⁴C]glucose.

Example 18. Comparison of stimulation of glucose uptake and metabolism in mouse embryo NIH 3T3 fibroblasts by PALP and Ins.

Data shown in Fig. 14 is for an experiment comparing the effects of PALP and Ins on glucose metabolism in mouse embryo NIH 3T3 fibroblasts. This cell line was chosen because it is relatively insensitive to Ins, and, therefore, it appeared to be suitable to further verify what had been suggested by animal experiments; namely, that the effects of PALP occur independently of Ins. Since serum may contain Ins, the fibroblasts were incubated in serum-free medium to further exclude any contribution by Ins.
Accordingly, confluent NIH 3T3 fibroblast cultures in 12-well plates were incubated for 5 hours in serum-free glucose-free DMEM with 1 µCi/mL of D-[¹⁴C]glucose in the absence (□) or presence of 1 U/mL commercial PALP
, 50 nM Ins
, or 1 U/mL commercial PALP + 50 nM Ins (■). The data shown in Fig. 14 are the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results).
PALP enhanced cellular uptake of D-[¹⁴C]glucose by38% and its metabolism to [¹⁴C]glycogen and [¹⁴C]total lipid by 77% and 55%, respectively. In each case, treatment with Ins alone
was only about 50% as effective as PALP alone
, and Ins did not enhance any of the effects of PALP when used in combination (■), relative to treatment by PALP alone
. Thus, this experiment demonstrates that PALP exerts an effect on glucose metabolism without requiring the presence of insulin.

Examples 19 and 20 - Effects of PALP preparations on glucose metabolism in differentiated L1 cells in the presence of 10% serum.

In the presence of 10% FBS most cellular events, particularly cell proliferation, are maximally or near maximally stimulated due to the presence of optimum concentrations of growth factors. It is difficult to assess which serum level (2% or 10% serum) used for in vitro experiments corresponds better to the situation in vivo (which, in turn, will also depend on the eating habit and the combination of various physiological parameters of the individual). However, as a conservative approach, it is generally accepted that if an effect can be observed in vitro even in the presence of 10% serum, then it is very likely that this particular phenomenon is physiologically relevant. Therefore, next it was determined if PALP and Ins still can enhance glucose metabolism in the presence of 10% serum, and if the effects of preparations made from various batches of commercial PALP were comparable.

Example 19. Effect of PALP preparations and Ins on glucose content of incubation medium for differentiated L1 cells.

Data shown in Fig.15 is for an experiment in which the effects of four different preparations made from various batches of commercially obtained PALP- on the loss of D-[¹⁴C]glucose from the incubation medium in differentiated L1 cell cultures were compared to the effect of Ins.
Preparations were made from four separate batches of commercially obtained PALP- (preparations 1 through 4), the preparations comprising 1.5 U/mL PALP in serum-free glucose-free DMEM. Differentiated L1 cells were pretreated for 30 minutes with PALP-free DMEM
, with preparation 1
, 2
, 3
, or 4
, or with 50 nM Ins in serum-free glucose-free DMEM (■), followed by the addition of 0.5 µCi of D-[¹⁴C]glucose for 10 hours. The data shown in Fig. 15 are [¹⁴C]glucose content of the incubation medium at the end of incubation (time = 10 hours), compared to [¹⁴C]glucose content at the start of incubation (time = 0 hours; □), and represent the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results).
While all four PALP preparations clearly enhanced the loss of D-[¹⁴C]glucose from the medium, there was some variability in their respective effects which appeared to go beyond the experimental error. Again, 50 nM Ins was more effective than any of the commercial PALP preparations.

Example 20. Exploration of the differences in effect on glucose metabolism for various PALP preparations.

Data shown in Fig. 16 is for an experiment in which potential differences in the effects on glucose metabolism in differentiated L1 cells among the four commercial PALP preparations was further explored. Differentiated L1 cells were pretreated (in 10% FBS-containing medium) for 30 min with PALP-free DMEM (□), or with preparation 1
, 2
, 3
, or 4
of commercial PALP (1.5 U/mL), or 50 nM Ins in serum-free glucose-free DMEM (■), followed by the addition of 0.5 µCi of D-[¹⁴C]glucose for 6 hours. The data are the mean ± S.D. of three determinations in one experiment (the experiment was repeated once with similar results).
While, again, each PALP preparation enhanced the synthesis of both [¹⁴C]glycogen and [¹⁴C]totallipid, the preparation which decreased the medium content of D-[¹⁴C]glucose the most (PALP 1 in Fig. 15) was also the most effective in stimulating the synthesis of [¹⁴C]glycogen and [¹⁴C]total lipid (Fig. 16).
In addition to demonstrating variability in the actions of various batches of commercially obtained PALP, the data in Figs. 15 and 16 also indicate that PALP exerts considerable effects on glucose metabolism in L1 cells even in the presence of 10 % serum. Such effects of PALP were observed in the presence of 10% serum only if cells were used 8-12 days after terminating the differentiation treatment.

Example 21 and 22 - Effect of homogeneous purified PALP on glucose metabolism in differentiated L1 cells.

In the experiments described in Examples 21 and 22, homogeneous purified PALP was used to demonstrate that the effects of partially purified commercial PALP that were observed in preceding Examples were indeed elicited by activity of the PAL-enzyme and not by a contaminating protein.
Homogeneous purified PALP- was prepared by the complete purification procedure described in Example 1. The homogeneous purified PALP used in the experiments of Examples 21 and 22 produces a single band in an electrophoretic separation, such as shown in lane 5 of Fig. 1.

Example 21. Effect of homogeneous purified PALP on glucose content of incubation medium in differentiated L1 cells.

Data for this Example is shown in Fig. 17. Differentiated L1 cells in 12-well plates were incubated in 10% serum-containing glucose-free DMEM for 10 hours with 0.5 µCi of D-[¹⁴C]glucose in the absence
or presence of 200 nM homogeneous purified PALP
, or 50 nM Ins (■), followed by the determination of D-[¹⁴C]glucose in the medium. The data shown in Fig. 17 are [¹⁴C]glucose content of the incubation medium at the end of incubation (time = 10 hours), compared to [¹⁴C]glucose content at the start of incubation (time = 0 hours; □), and represent the mean ± S.D. of three determinations in one experiment.
Homogeneous purified PALP at 200 nM effectively reduced the medium content of D-[¹⁴C]glucose, although its effect was somewhat less than that of 50 nM insulin. The effects of purified PALP on glucose uptake were comparable to the effects of the best commercial PALP preparation demonstrated in Example 19 (Fig. 15).

Example 22. Effect of homogeneous purified PALP on glucose uptake and metabolism for differentiated L1 cells.

Data for this Example is shown in Fig. 18. Homogeneous PALP was used to demonstrate that the observed effects of PALP on the cellular uptake and metabolism of D-[¹⁴C]glucose from prior Examples was indeed mediated by the PALP enzyme. Differentiated L1 cells in 12-well plates were incubated in 10% serum-containing glucose-free DMEM for 6 hours with 0.5 µCi of D-[¹⁴C]glucose in the absence (□) or presence of 200 nM homogeneous purified PALP
, or 50 nM Ins (■). The data are the mean ± S.D. of three determinations in one experiment.
Homogeneous PALP increased cellular content of D-[¹⁴C]glucose 1.4-fold, increased synthesis of [¹⁴C]glycogen 1.95-fold, and increased [¹⁴C]total lipid 2.45-fold. Again, the effects of homogeneous purified PALP were comparable to the effects of the less pure commercial PALP preparations demonstrated in Example 20 (Fig. 16). Overall, it seems clear that the reproducible effects of PALP are the best assured if PALP is purified to homogeneity.

Example 23. Effect of α-antitrypsin on glucose levels in the glucose tolerance test.

α-antitrypsin is the major contaminant of the commercially obtained PALP [Q.B.-She, J.J. Mukherjee, K.S. Crilly, and Z. Kiss, "α-antitrypsin can increase insulin-induced mitogenesis in various fibroblast and epithelial cell lines." FEBS Letters 473, 33-36 (2000)]. Therefore, an experiment was performed to determine whether α-antitrypsin has PALP-like effects on blood glucose levels. A glucose tolerance test was performed as generally described in Example 2. In the experiment, highly purified α-antitrypsin was administered either intraperitoneally or subcutaneously at a quantity of 2 mg/mouse, 24 hours prior to administration of glucose.
α-antitrypsin failed to lower the blood glucose level (data not shown). This observation indicates that α-antitrypsin did not mediate the effect on blood glucose levels that were observed for administration of commercial PALP.

Example 24. PALP-antibody blocks the effects of homogeneous purified PALP on glucose metabolism.

Even the homogeneous purified PALP preparation used in Examples 21 and 22 still might have contained a very small amount of an active contaminant which escaped detection. To rule out this possibility, a PALP-specific antibody (polyclonal rabbit anti-PALP from Zymed Laboratories, South San Francisco, California) was used to block the effect of the PALP enzyme.
Data from the experiment is shown in Fig. 19. Differentiated L1 cells in 12-well plates were incubated in 10% serum-containing glucose-free DMEM for 6 hours in the absence (□) or presence of 30 µg/mL of PALP-antibody
, 15 µg/mL of homogeneous purified PALP
, or 30 µg/mL of PALP-antibody + 15 µg/mL of homogeneous purified PALP (■). The data are the mean ± S.D. of three independent determinations in one experiment.
While PALP-antibody alone had no effect on glucose metabolism, PALP-antibody clearly blocked the stimulatory effects of homogeneous purified PALP on the uptake of D-[¹⁴C]glucose, as well as the synthesis of [¹⁴C]glycogen and [¹⁴C]total lipid. Thus, this experiment provides conclusive proof that the effects on glucose metabolism observed with commercial PALP preparations and homogeneous purified PALP preparations were indeed mediated by the PALP enzyme.
Based on these data, the conclusion can be made that in the reported in vivo experiments (Examples 2-8), the inhibitory effects of commercial PALP on the elevation of blood glucose level were mediated by the PALP enzyme. The present invention may thus provide an effective newtreatment for diabetes that would meet each criterion listed above. In particular, the experiments presented in the Examples indicate that treatment regimens including the administration of PALP would not cause weight gain and could diminish weight loss. Furthermore, the experiments presented indicate that the active agent (PALP) acts via mechanism(s) that are independent from the actions of insulin, and that the active agent is metabolically stable, which permits less frequent usage. Finally, the results suggest that PALP may prolong the life of severely diabetic patients.
The invention described herein includes purified human placental alkaline phosphatase for use in a medicament. The invention further includes purified human placental alkaline phosphatase for use in a medicament for the treatment of diabetes. The invention includes the use of purified human placental alkaline phosphatase for the manufacture of a medicament for the treatment of diabetes. The invention further includes use of purified human placental alkaline phosphatase for the manufacture of a medicament for reducing blood glucose level. Also included as part of the invention is the use of purified human placental alkaline phosphatase for the manufacture of a medicament for reducing weight gain or inducing weight loss.

Claims

Purified human PALP (placental alkaline phosphatase), or a physiologically active derivative thereof, for use in the treatment of diabetes, whereby the derivative is selected from fragments of PALP that demonstrate efficacies similar or more effective than native PALP.
Purified human PALP, or a physiologically active derivative thereof, for use in reducing plasma glucose levels whereby the derivative is selected from fragments of PALP that demonstrate efficacies similar or more effective than native PALP.
Purified human PALP, or a physiologically active derivative thereof, and an anti-diabetic medicament for use in the treatment of diabetes, wherein human placental alkaline phosphatase, or a physiologically active derivative thereof, and the anti-diabetic medicament are to be used simultaneously, separately or sequential, whereby the derivative is selected from fragments of PALP that demonstrate efficacies similar or more effective than native PALP.
Purified human PALP and anti-diabetic medicament for use according to claim 3, wherein the anti-diabetic medicament is an insulin secretogogue, a biguanide, an inhibitor of α-glucosidase, a thiazolidinedione, NN2211, or insulin.
PALP or a physiologically active derivative thereof for use according to claims 1 or 2, wherein the active derivative is a protease digestion product of human PALP, whereby the derivative is selected from fragments of PALP that demonstrate efficacies similar or more effective than native PALP.
PALP or a physiologically active derivative thereof for use according to claim 5 wherein the protease is bromelain.
Purified human PALP or a physiologically active derivative thereof for use according to claim 1 or 2 or purified human PALP or a physiologically active derivative thereof and an anti-diabetic medicament for use according to claim 3 in the form of a preparation together with a physiological saline solution as a carrier.
Purified human PALP or a physiologically active derivative thereof or purified human PALP or a physiologically active derivative thereof and an anti-diabetic medicament for use according to claim 7 wherein the preparation includes human PALP enclosed within liposomes.
Purified human PALP or a physiologically active derivative thereof for use according to claim 1 or 2 or purified human PALP or a physiologically active derivative thereof and an anti-diabetic medicament for use according to claim 3 wherein the human PALP is tissue-extracted.
Purified human PALP or a physiologically active derivative thereof for use according to claim 1 or 2 or purified human PALP or a physiologically active derivative thereof and an anti-diabetic medicament for use according to claim 3 wherein the human PALP is recombinant-derived human PALP.
Purified human PALP or a physiologically active derivative thereof for use according to claim 1 or 2 or purified human PALP or a physiologically active derivative thereof and an antidiabetic medicament for use according to claim 3 wherein the human PALP is synthetic human PALP.